Material and methods

Plant: The stem bark of the plant Lantana indica and Alstonia scholaris were collected from the botanical garden of DDU, Gorakhpur University, Gorakhpur, Uttar Pradesh, India. First of all, stem bark of both the plants were washed separately with water and then dried in an incubator at about 37⁰ C. Then dried stem bark of Lantana indica and Alstonia scholaris were powdered with the help of mechanical device.

Extraction of compounds: 300 gram powder of stem bark of Lantana indica or Alstonia scholaris were subjected to extraction through Soxhlet apparatus in methanol solvent for about 50 hours and a concentrated solution was obtained. After evaporation of solvent, the extracted compound in dried form was obtained. This extracted compound was stored in air-tight desicator and further used for experiments.

Animal: The fresh water harmful snail Lymnaea acuminata (3.65± 1.00 cm total shell height and 1.40 ± 0.50 cm total shell width), were collected from the fresh water bodies of Gorakhpur. Prior to experiment snails were allowed to acclimatize for 72h.

Toxicity experiment: The experiment was conducted in freshwater ponds, 29.28 m³ in area and 9.19 m³ in water volume. Each pond was stocked with 100 snails with a size difference not greater than 1.5 times (APHA, 1992). The experimental animals were exposed continuously 24h up to for 96h to four different concentrations. Control groups were kept under similar conditions without any treatment. Mortality was recorded after every 24h during the observation period of 96h.  Contraction of the snail body within the shell and no response to a needle probe were taken as evidence of death of snails. Dead animals were removed to prevent the decomposition of body in pond.

 Lethal concentration (LC₅₀) values for different exposure periods, lower confidence limits (LCL) and upper confidence limits (UCL), slope value,‘t’ ratio and heterogeneity were calculated by using probit log analysis of POLO Plus computer programme of Robertson et al., 2007.

Biochemical Estimations:

The biochemical experiments were performed by the method of Tripathi and Singh (2001). The biochemical experiment was conducted in freshwater ponds. Each pond was stocked with 100 snails in 29.28 m³ in area and 9.19 m³ in water volume with a size difference not greater than 1.5 times (APHA, 1992). The experimental animals were treated with two different sub-lethal doses, i.e. 40% and 80% of LC₅₀ (24h) of methanol extract of stem bark of Lantana indica or Alstonia scholaris singly or in binary mixtures for 96h. Control groups were kept under similar conditions without treatment for same duration. Diet was not given to the snail during the course of experiment. After the completion of 96h of treatment snails were removed from the treated water and the nervous and hepatopancreas tissues of both the treated as well as control group were quickly dissect out and used for biochemical estimations.

In order to see the effect of 7^th day of withdrawal, animals were exposed to sub lethal doses i.e. 80% of LC₅₀ of 24h singly i.e. Lantana indica bark methanol extract (LIBME) or Alstonia scholaris bark methanol extract (ASBME) or in binary mixture of LIBME+ASBME in 1:1 ratio for 96h exposure periods. After 96h animals were transferred to freshwater free from any treatment for the seven day. After completion of 7^th day the nervous and hepatopancreas tissue was quickly dissect out and used for bio-chemical parameters.

Total free amino acid: Estimation of total free amino acid was made according to the method of Spies (1957). Homogenates (50 mg/mL, w/v) were prepared in 95% ethanol, centrifuged at 6000 xg and used for amino acid estimation.

Protein: Protein levels were estimated according to the method of Lowry et al., 1951 using bovine serum albumin as standard. Homogenate (50 mg/mL, w/v) were prepared in 10% TCA.

Glycogen: Glycogen was estimated by the Anthrone method of Vander Vies 1954, as modified by Mahendru and Agarwal 1982. 50 mg of tissue were homogenised with 5 mL of cold 5% TCA. The homogenate was filtered and 1.0 mL of filtrate was used for assay.

Nucleic acid: Estimation of nucleic acid (DNA and RNA) performed by the method of Schneider (1957), using diphenylamine and orcinol reagents, respectively. Homogenates (1 mg/mL, w/v) were prepared in 5% TCA and centrifuged at 5000 xg for 20 minute and supernatant was prepared and used for estimation.

Activity of enzyme protease: Protease activity was measured according to the method of Moore and Stein (1954).  Homogenate (50 mg/L, w/v) was prepared in cold distilled water (0^o C) and optical density was measured at 570 nm. Protease activity is expressed in as micromoles of tyrosine equivalents/mg protein /h.